Rotaviruses are the primary cause of severe dehydrating diarrhea in infants and young children, globally causing an estimated 2 million hospitalizations and 440,000 deaths per year in children under the age of 5. Due to its significant morbidity and mortality, an important goal of the Laboratory of Infectious Diseases remains the development of methods for controlling, preventing, and treating rotaviral disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of rotavirus, notably those events in the viral life cycle connected to the replication of the virus's segmented double-stranded (ds)RNA genome. An expected outcome of this project is the development of a reverse genetics system that could be used in manipulating the genetic information of the virus. Such a system would provide an important tool for generating new and modifying existing rotavirus vaccines. This project may also lead to the identification of potential targets for antiviral components that can subvert the rotavirus replication cycle. Specifically, this project seeks to identify and characterize molecular signals in viral RNAs that function in the expression of rotavirus genes and in the packaging and replication of the rotavirus genome. The project also seeks to characterize the structure and function of those viral proteins involved in these processes. These aims will be accomplished by a combination of procedures, which include (i) analysis of the replication and translation efficiencies of mutated viral mRNAs in cell-free systems, (ii) computer modeling and structural analysis (e.g., RNAse mapping, NMR spectroscopy) of the recognition signals in the mRNAs, (iii) characterization of the enzymatic and structural properties (e.g., analytical ultracentrifugation, X-ray crystallography, cryo-electron microscopy, CD spectroscopy) of recombinant viral proteins, and (iv) elucidation of the specificity and targets of the viral RNA-binding proteins by gel mobility shift assay and RNA-protein cross-linking. Studies performed in the last year have provided insight into (i) the importance of viral replication factories (viroplasms) as sites of RNA synthesis in infected cells, (ii) the enzymatic properties of NSP2, a viral protein required for viroplasm formation and involved in genome packaging, and (iii) the role of the conserved ends of viral RNAs in genome replication and gene expression. Collectively, these studies have been particularly significant in suggesting new avenues for achieving a reverse genetics system for the rotaviruses, and in providing possible explanations for the failures of earlier attempts.